CN109596837B

CN109596837B - Bionic digestion determination method for protein digestibility of pig feed

Info

Publication number: CN109596837B
Application number: CN201811503672.9A
Authority: CN
Inventors: 赵峰; 王亚; 张虎; 高庆涛
Original assignee: Institute of Animal Science of CAAS
Current assignee: Institute of Animal Science of CAAS
Priority date: 2018-12-10
Filing date: 2018-12-10
Publication date: 2022-02-08
Anticipated expiration: 2038-12-10
Also published as: CN109596837A

Abstract

The invention discloses an in-vitro bionic digestion method for rapidly determining the digestibility of pig feed protein, which comprises the following steps: crushing the feed sample and screening the crushed feed sample by a standard sieve; preparing a gastric buffer solution and a small intestine buffer solution; preparing simulated gastric juice, concentrating simulated small intestinal juice and concentrating and supplementing small intestinal juice; filling the stomach buffer solution and the small intestine buffer solution into a heat preservation chamber for bionic digestion of monogastric animals, and connecting a pipeline communicated with a simulated digester; loading the crushed feed sample into a simulated digester, adding simulated gastric juice, and loading into a bionic digestive system of a monogastric animal; automatically completing the in-vitro bionic digestion process of the pig feed protein by setting the bionic digestion parameters of the stomach and the small intestine at each stage through control software of a bionic digestion system; and after obtaining undigested residues, drying, determining the crude protein content of the residues and feed samples, and calculating the digestibility of the protein. The method is simple, high in precision and low in implementation cost, and can accurately measure the protein digestibility of the pig feed within 48 hours.

Description

Bionic digestion determination method for protein digestibility of pig feed

Technical Field

The invention relates to a bionic digestion determination method for the digestibility of pig feed protein, in particular to a method for estimating the digestibility of the feed protein by fully automatically simulating the in-vivo digestion process of pigs in vitro under the laboratory condition.

Background

The contents of digestible protein, amino acid and other nutrients of the feed are main decision bases for guiding enterprises to establish a feed raw material protein titer database and a formula standard, and to bid purchase and optimize the feed formula, and are also important technical supports for reducing the nitrogen excretion of livestock and poultry from the source in China. At present, in the production of compound feed, the data is not actually measured data, but table look-up or experience correction is carried out through crude protein content, so that larger deviation and misleading exist, and the data becomes the first recessive waste of feed industry. For nearly half a century, standard ileal terminal (SID) digestible crude protein or amino acid data were obtained by metabolic testing after mounting a cannula at the pig ileal terminal, on a method for measuring digestibility of feed proteins or amino acids. The animal test method has the advantages that the determination period is 10-15 days, the time consumption is long, the system error is large, the precision is low, the repeatability is poor and the like, and the basic requirements of modern animal husbandry on accurately and quickly obtaining the digestible crude protein or amino acid content of the feed cannot be met.

In order to meet the requirement that the digestibility of the protein or the amino acid of the pig feed can be rapidly determined under the laboratory condition in the enterprise production, developed countries in Europe, America and the like are always dedicated to establishing a technology for rapidly evaluating the content of the digestible protein and the amino acid of the feed by simulating the digestion of the pig feed in the stomach-small intestine. The technical rules for rapid assessment of protein digestibility of pig feed based on trypsin, a system established by Boisen and Fernandez, by Danish scholars, are relatively widely applied internationally (see references: Boisen, S., and J.A.Fernandez. prediction of the topical systemic differentiation of proteins and amino acids in feedback and feedback for pigs by in vitro analysis, animal. feed Sci.Technol.,1995,51: 29-43). However, this method has the following drawbacks: 1) in the preparation of simulated digestive juice of stomach and small intestine, the activity of digestive enzyme in pancreatin which is a reagent used for preparing the simulated digestive juice is unclear, so that the activity of the prepared simulated digestive juice is also unclear, and the digestive ability is difficult to repeat; 2) in the process of simulating digestion, the composition of digestive juice in the pig body is not clarified, so that the activity of enzymes in the digestive juice in the digestion stage of the stomach and the small intestine is greatly different from that in the body on the premise of unclear simulation target objects; 3) in the simulated digestion process of the small intestine of the pig, the simulated small intestine liquid is added once when the small intestine digestion is started, and the activity of the digestive enzyme is stable or attenuated in the digestion process is unclear. Since the activities of pancreatic juice secretion and digestive tract digestive enzyme are periodically changed with ingestion, the digestive enzyme activity decays in vivo, but the activity of the digestive enzyme changes in a pulse form due to the secretion of pancreatic juice. How to simulate the phenomenon in-vitro digestion is still rarely reported; 4) the in vitro simulated digestion process is carried out in a closed system taking a triangular flask as a reaction container, and the change of the pH value, the addition of digestion liquid, the separation of products and the like in each digestion stage are realized by manual operation; 5) in the separation of hydrolysate, undigested protein is precipitated by sulfosalicylic acid, and hydrolysate and unhydrolyzed product are separated by filtration, so that the undigested protein is often filtered out and regarded as digested protein, and the measured value is greatly higher than the measured value in vivo. Due to the above problems, the method is difficult to standardize in many aspects, so that the method is not adopted in feed industry for measuring the protein titer of feed raw materials in developed countries such as the United states, Canada and the like and China.

In recent years, in order to eliminate the interference of manual operation on the in vitro simulated digestion test result, a monogastric animal bionic digestion system special for pigs and poultry was developed in 2009 by the Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences in combination with the modern automatic control technology (see the Chinese invention patent 'monogastric animal bionic digestion system and a method for simulating monogastric animal digestion based on the system' with the patent number ZL 200910078147.1). At present, the patented technology is assigned, and standardized monogastric animal bionic digestion instrument products are produced for industry use (purchased by domestic research units and feed and breeding enterprises). The instrument provides a technical basis for standardization of in vitro digestion technology of pigs and application of the instrument to rapid determination of contents of digestible protein and amino acid of feed. On the other hand, with the development of protein purification technology, professional companies (e.g., Sigma, Amersham) have produced highly pure reagent-grade digestive enzymes and supplied them to the outside. By taking the activity of the digestive enzyme as a reference and preparing the simulated digestive juice by using the reagent-grade digestive enzyme, the digestion activity of the digestive juice used in the in-vitro digestion can be standardized. Therefore, on the basis of clearing basic parameters of the digestion process in the pig body, the standardization of the simulated digestive juice and the automation of the digestion process in vitro are realized on the basis of a full-automatic digestion simulation tool and a high-purity reagent enzyme in vitro; on the basis of clearing the decay rule of the digestive enzyme activity in the in vitro simulated digestion process, the degree of in vitro simulated digestion on the body is further improved by supplementing the digestive enzyme. Based on the technical improvement, the defects of the existing in-vitro simulation digestion method can be overcome. In conclusion, the development of a new method is further approaching to the simulation of the digestion process in the pig body to rapidly measure the content of digestible protein and amino acid of the feed, and the important technical requirement of the industry is absolutely met.

Disclosure of Invention

The invention aims to provide an in vitro rapid determination method for the digestibility of protein or amino acid in pig feed. The method realizes accurate estimation of the pig feed protein or amino acid ileum terminal digestibility by in-vitro full-automatic simulation of digestion of two stages of pig stomach and small intestine, solves the defects of unclear simulated digestion process digestive enzyme activity, digestive enzyme activity attenuation, manual operation of the digestion process and over 10 percent difference from the in-vivo determination of the pig feed ileum terminal crude protein digestibility in the traditional method, and has the advantages of energy saving, resource saving, high repeatability and high testing speed.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for determining the digestibility of pig feed protein by in vitro biomimetic digestion comprises the following steps:

1) sample pretreatment: crushing a pig feed sample, sieving the crushed sample by a standard sieve, and then vacuumizing and filling nitrogen into the crushed sample and storing the crushed sample at a low temperature for later use;

2) preparation of a buffer: preparing a gastric buffer solution by using dilute hydrochloric acid, sodium chloride, potassium chloride and an antibacterial agent; preparing small intestine buffer solution by phosphate, sodium chloride, potassium chloride and an antibacterial agent;

3) preparation of simulated gastric fluid: preparing simulated gastric juice by using reagent-grade pepsin according to the activity of pepsin in gastric juice of pigs;

4) preparation of simulated small intestine solution: according to the activity of digestive enzyme in the porcine intestinal fluid, preparing concentrated simulated intestinal fluid by using reagent-grade alpha-amylase, trypsin and chymotrypsin; according to the decay condition of digestive enzymes in the middle and small intestine stage of in vitro simulated digestion, preparing concentrated supplementary simulated small intestine solution by using reagent-grade alpha-amylase, trypsin and chymotrypsin;

5) loading: the bionic digestion system for the monogastric animals comprises a simulated digester, a digestive juice reagent bottle, a buffer solution reagent bottle, a waste liquid containing bottle, a cleaning liquid storage bottle, a cleaning liquid reagent bottle and a cleaning residual liquid containing bottle, wherein the simulated digester comprises a glass tube and a dialysis tube, the glass tube is a hollow tube body, two ends of the tube body are respectively provided with a ground opening, the side surface of the tube body is provided with an input tube and an output tube, the dialysis tube is placed in the glass tube, two ends of the dialysis tube respectively extend out of the two ground openings of the glass tube and turn outwards, the end part of the dialysis tube which is turned outwards and exposed out of the ground opening is tied up and fixed on the ground opening by a rubber strip, and a silica gel plug with a liquid conveying tube are respectively plugged in the two ground openings after the end part of the dialysis tube is tied up;

putting the concentrated simulated small intestine solution obtained in the step 4) and the supplemented concentrated simulated small intestine solution into a digestive juice reagent bottle of the bionic digestive system of the monogastric animals; placing the gastric buffer solution and the small intestine buffer solution obtained in the step 2) into a buffer solution temperature control room of the bionic digestive system of the monogastric animals; after the monogastric bionic digestion system is operated for 30-60 minutes in advance and each temperature parameter reaches 39 ℃, pouring the feed sample prepared in the step 1) into a dialysis tube of a simulated digester of the monogastric bionic digestion system, adding the simulated gastric juice obtained in the step 3) into the dialysis tube, and plugging a rubber plug;

6) gastric simulated digestion: pumping the gastric buffer solution out of a dialysis tube of the simulated digester and then returning the gastric buffer solution into a gastric buffer solution reagent bottle for reciprocating circulation, and providing hybrid power of simulated gastric juice and a feed sample through rotary oscillation to perform simulated digestion of the stomach; discharging the gastric buffer solution in the simulated digester after digestion is finished, and pumping deionized water into the gastric buffer solution to clean hydrolysate remained in the dialysis tube in the simulated digestion stage of the stomach; discharging the cleaning liquid in the simulated digester after the cleaning is finished; the process is repeatedly cleaned for a plurality of times so as to be cleaned;

7) simulated digestion of the small intestine: pumping the small intestine buffer solution out of a dialysis tube of the monogastric animal simulated digester and returning the small intestine buffer solution to a small intestine buffer solution reagent bottle for reciprocating circulation, providing mixed power of concentrated simulated small intestine solution and a feed sample through rotary oscillation, and injecting the concentrated simulated small intestine solution into the dialysis tube after the pH value of the solution in the dialysis tube is changed into the pH value of the small intestine buffer solution to start simulated digestion of the small intestine; injecting concentrated supplementary simulated small intestine solution into the dialysis tube again after digestion for 4h, and continuing small intestine simulated digestion; discharging the buffer solution in the simulated digester after digestion is finished;

8) cleaning of hydrolysate: pumping deionized water out of a dialysis tube of the simulated digester to clean hydrolysate remained in the dialysis tube in the whole simulated digestion stage; discharging the cleaning liquid in the simulated digester after the cleaning is finished; repeated for many times so as to clean thoroughly;

9) analysis of undigested residue and calculation of digestibility: transferring the residue remained in the dialysis tube after the cleaning in the step 8) to a culture dish, drying, respectively measuring the crude protein or amino acid content of the residue and the feed sample, and calculating the digestibility of the crude protein or amino acid.

In the step 1), the pig feed is a single feed raw material or a compound feed, such as corn type feed, soybean meal type feed, wheat bran type feed, corn-soybean meal type feed and the like.

The comminution is carried out in a universal mill. When the feed raw materials are crushed into 2mm sieve pieces in production, crushing the samples in a universal crusher for 20-60 s, and sieving the samples with a 40-mesh grading sieve; when the feed raw materials are crushed and sieved by a sieve sheet with the thickness of 1mm in production, the sample is crushed in a universal crusher for 60-90 s and sieved by a 60-mesh classifying sieve.

In the step 1), the method for performing vacuum-nitrogen treatment on the crushed sample specifically comprises the following steps: filling the crushed sample into a breathable cotton paper bag, sealing the bag, then sleeving the bag into a plastic sample bag, adjusting the air suction time to be 10-20 s, the air inflation time to be 2-4 s and the heating sealing time to be 1.5-4.5 s on a vacuum packaging machine, and then carrying out vacuumizing-nitrogen filling-sealing treatment. The low-temperature storage temperature is-20 to-10 ℃.

In the step 2), the concentration of hydrochloric acid in the gastric buffer solution is 5-10 mmol/L, the concentration of NaCl is 60-110 mmol/L, the concentration of KCl is 4-8 mmol/L, and the concentration of an antibacterial agent is 1-2 g/L; adjusting the pH value to 2-3 at 39 ℃; the antimicrobial agent may be potassium sorbate.

In the step 2), the concentration of disodium hydrogen phosphate in the small intestine buffer solution is 30mmol/L, the concentration of sodium dihydrogen phosphate is 170mmol/L, the concentration of NaCl is 70-106 mmol/L, the concentration of KCl is 14-17 mmol/L, the concentration of penicillin is 800U/L, and the concentration of an antibacterial agent is 1-3 g/L; adjusting the pH value to 6.4-6.5 by using 1-2 mol/L phosphoric acid or sodium hydroxide at 39 ℃; the antimicrobial agent may be potassium sorbate.

In the step 3), the activity of pepsin in the simulated gastric juice is 737-1000U/mL, the concentration of hydrochloric acid is 5-10 mmol/L, the concentration of NaCl is 60-110 mmol/L, the concentration of KCl is 4-8 mmol/L, and the pH value is 2-3.

In the step 4), the concentration of amylase in the concentrated simulated small intestine solution is 2046-2893U/mL, the concentration of trypsin is 627-858U/mL, and the concentration of chymotrypsin is 77-110U/mL.

The concentration of amylase in the concentrated supplementary simulated small intestine solution is 1935-2139U/mL, the concentration of trypsin is 1359-1502U/mL, and the concentration of chymotrypsin is 48-53U/mL.

Pepsin used in the present invention is specifically available from Sigma under catalog number P7000; amylases are specifically available from Sigma, usa under catalog number a 3306; trypsin is specifically available from Amersham corporation under product catalog number 0785; chymotrypsin is specifically available from Amershamo under catalog number 0164.

In the step 5), the monogastric bionic digestion system is a monogastric bionic digestion system described in chinese patent ZL 200910078147.1, and specifically may be a monogastric bionic digestion system of model SDS-2 produced by smart technology limited in south of Hunan. The cut-off molecular weight of the dialysis tube is 12000-14400 daltons, and the flat diameter is 44 mm; the opening volume of the tube body is 35-45 mL.

The sample loading amount of each simulated digester in the bionic digestive system of the monogastric animals is 1-2 g, the volume of simulated gastric juice is 20mL, the dosage of concentrated simulated small intestinal juice is 2mL, the dosage of concentrated supplemented small intestinal juice is 1mL, the dosage of gastric buffer is 200mL, and the dosage of small intestinal buffer is 200 mL.

In the step 6), the digestion temperature is 39 ℃; the rotary oscillation frequency of the feed sample mixed with the simulated gastric juice is 180 revolutions per minute; the flow rate of the buffer solution flowing through the simulated digester was 60 mL/min; the simulated digestion time of the stomach is 4 hours; after the simulated digestion of the stomach is finished, the cleaning liquid amount for cleaning the digestion product at one time is 300mL of deionized water/simulated digester, each time the digestion product is cleaned for 40 minutes, and the cleaning is repeated for 3 times.

In the step 7), the digestion temperature is 39 ℃; the convolution oscillation frequency of the sample mixed with the concentrated simulated small intestine solution is 180 r/min; the flow rate of the buffer solution flowing through the simulated digester was 60 mL/min; the small intestine simulates digestion for 8h, wherein the supplementary concentrated simulated small intestine fluid is injected 4h after the small intestine simulates digestion and continues digestion for 4 h.

In the step 8), the cleaning liquid amount for cleaning the digestion product at one time after the whole simulated digestion is finished is 300mL of deionized water/simulated digester, the cleaning is carried out for 2-4 h every time, and the cleaning is repeated for 6 times.

In the step 9), the undigested residues are transferred into a culture dish with constant weight, and then are dried in a constant temperature air blast drying oven at 65 ℃ until no water mark exists, or are freeze-dried and then are dried in a constant temperature air blast drying oven at 105 ℃ until the weight is constant.

In the step 9), the feed sample and the undigested residue are subjected to nitrogen fixation by a Kjeldahl method to determine the content of crude protein, and the digestibility of the protein is calculated. The content of the amino acid can also be measured by a liquid chromatogram or a full-automatic amino acid analyzer, and the digestibility of the amino acid can also be calculated.

Compared with the prior art, the invention has the following advantages:

1. aiming at the physiological phenomenon that the digestion rates are different due to different chyme particle sizes of feed raw materials with different crushing particle sizes when the pigs digest in the production and processing of compound feed, the method establishes that in the bionic digestion process, the ground sample passes through a 40-mesh classifying screen and corresponds to the ground chyme particle size in the pig body which passes through a 2mm sieve in the actual production, and the sample passes through a 60-mesh classifying screen and corresponds to the ground chyme particle size in the pig body which passes through a 1mm sieve in the actual production. The granularity of the sample in the in vitro digestion process is corresponding to the granularity of the chyme in the pig body;

2. the quality guarantee process of the vacuumized-nitrogen-filled low-temperature storage sample is established, and the error caused by oxidative deterioration of the sample in the implementation process of the method is solved;

3. antibacterial and antifungal agents are added into the buffer solution, so that the degree of influence on bionic digestion due to the reduction of the pH value caused by microbial fermentation in the bionic digestion process is reduced to the maximum extent;

4. in the bionic digestion process of the stomach and the small intestine, the activity of digestive enzymes and the ion concentration of a buffer solution in a solution after the feed is mixed with a digestive juice are kept consistent with those of a digestive juice in a pig body, the activity concentration of the digestive enzymes influencing the digestion degree is determined on the preparation of the digestive juice to be used as the preparation basis, and the defects that the activity of the simulated digestive juice is unclear and can not be repeated because the weight of a digestive enzyme reagent is used as the preparation basis in the traditional method are overcome;

5. after the concentrated simulated small intestine digestive juice is pumped in the small intestine digestion stage, the active concentration of the main digestive enzyme in the digestive juice in the dialysis tube is consistent with the active concentration of the corresponding digestive enzyme in the pig small intestine juice. The problem that the digestive enzyme activity is far lower than the digestive enzyme activity concentration of intestinal juice in vivo due to poor solubility of pancreatin after the pancreatin solution is added in the digestive stage of small intestine in the traditional method is solved;

6. according to the attenuation change condition of digestive enzymes in the small intestine stage in bionic digestion, the concentrated small intestine solution is supplemented once in the small intestine digestion stage, so that the pulse change of the activity of the digestive enzymes in the small intestine is achieved, and the change rule similar to that in a pig body is realized. Further reducing the difference between the in vitro and in vivo measurements.

7. In the bionic digestion process, low molecular substances formed by enzyme digestion are permeated into a buffer solution or deionized water for cleaning through the aperture of a dialysis tube, so that the absorption process in a pig body is simulated, the inhibition of the product on the digestion degree in vitro is reduced, and the approach degree of an in vitro measured value and an in vivo measured value is improved;

8. in the bionic digestion of the stomach and the small intestine, the pumping of buffer solution and digestive juice, the discharge of residual liquid, the switching from the digestion stage of the stomach to the digestion stage of the small intestine and the separation and cleaning of hydrolysate are all carried out by computer program control, thereby greatly reducing the accumulated error caused by the manual operation of each step in the traditional method and leading the coefficient of variation of repeated measurement to be lower than 1 percent.

Drawings

FIG. 1 is a flow chart of an implementation of the assay method provided by the present invention.

FIG. 2 is a graph of the activity of digestive enzymes in maize in the small intestine simulated digestion stage as a function of digestion time.

FIG. 3 is a graph of the digestive enzyme activity of soybean meal as a function of digestion time at the simulated digestion stage of the small intestine.

FIG. 4 is a graph of wheat bran digestive enzyme activity versus digestion time at the simulated digestion stage of the small intestine.

Fig. 5 is a graph of digestive enzyme activity versus digestion time for a corn-soybean meal type diet at the simulated digestion stage of the small intestine.

Detailed Description

The method of the present invention is illustrated by the following specific examples, but the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included within the scope of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Pepsin used in the examples below was purchased from Sigma under catalog number P7000; amylase was purchased from Sigma, usa under catalog number a 3306; trypsin is specifically available from Amersham corporation under product catalog number 0785; chymotrypsin is specifically available from Amershamo under catalog number 0164.

Example I Change of digestive enzyme Activity with digestion time in Swine stomach-Small intestine Bionical digestion Process and Effect of digestive enzyme supplementation in Small intestine digestion stage

1 feed raw material, daily ration and pretreatment

Corn (8.57% of crude protein, 1.36% of crude fiber and 3.03% of crude fat), soybean meal (42.50% of crude protein, 6.78% of crude fiber and 3.22% of crude fat), and wheat bran (16.15% of crude protein, 9.01% of crude fiber and 3.92% of crude fat) are randomly selected from the feed raw material market in China as representative samples. And corn-soybean meal type feed (comprising 67.03% of corn, 23.22% of soybean meal, 3.20% of wheat bran, 2.98% of soybean oil, 3.57% of multi-vitamin multi-mineral, 17.43% of crude protein, 2.90% of crude fiber and 4.80% of crude fat) is prepared. And (3) respectively crushing the 4 samples by using a universal crusher, then passing through a standard sieve hole of 60 meshes, respectively filling the crushed samples into breathable cotton paper bags, sealing the cotton paper bag openings on a thermoplastic sealing machine, and filling the cotton paper bags into plastic sample bags. A multifunctional vacuum packaging machine (Shandong City American food machinery factory, model DZ500) is adopted, the air extraction time is adjusted to 15s, the air inflation time is adjusted to 2.5s, the heating sealing time is adjusted to 1.8s, the sample bag is sealed by the vacuum-extraction nitrogen-filling procedure, and then the sample bag is stored at the temperature of minus 20 ℃ for standby.

2 design of the experiment

In the process of bionic digestion of pig stomach-small intestine, the change of digestive enzyme activity along with digestion time is examined after the simulated gastric juice and the simulated small intestine juice are injected into the feed of corn, soybean meal, wheat bran and corn-soybean meal in two stages of bionic digestion of pig stomach-small intestine at one time. Pepsin activity (specific activity per unit volume and total activity) was analyzed by taking a one-way completely random design and canceling the digestion solution samples after 0, 1, 2, 3, 4h of digestion at the gastric digestion stage. In the small intestine digestion stage, after digestion for 4 hours in the stomach, digestion-simulated small intestine fluid samples are injected for 0, 2, 4, 6, 8, 12 and 16 hours, and the activities of trypsin, chymotrypsin and amylase are analyzed. Each treatment was 5 replicates, each replicate 1 digestive tube. The effect of supplementing digestive enzymes in the small intestine digestion stage is that the digestive enzymes are supplemented when the digestion is carried out for 4 hours in the small intestine stage according to the rule of the activity change of the digestive enzymes in the previous test. The change of the digestive enzyme activity along with the digestion time in the bionic digestion of the small intestine of the corn, soybean meal, wheat bran and corn-soybean meal type feed for 4h, 6h and 8h is examined. Each treatment was 5 replicates, each replicate 1 digestive tube.

3 bionic digestion method of pig

Cutting a dialysis tube (with the molecular weight cutoff of 14000 daltons and the flat diameter of 44mm) into small sections of about 22-28 cm, and putting the small sections into a mixed solution of sodium bicarbonate with the concentration of 2% (W/V) and disodium ethylene diamine tetraacetate with the concentration of 1mmol/L and the pH value of 8.0. After heating to boiling on an electric ceramic furnace, slight boiling is kept for 10 minutes. And after the mixed solution is poured out, cleaning the dialysis bag for 3-5 times by using distilled water. Then, a solution of disodium ethylenediaminetetraacetate (1 mmol/L, pH 8.0) was added thereto, and boiled in an electric ceramic oven for 10 minutes. After the waste liquid was discarded, deionized water was added and stored in a refrigerator at 4 ℃ for further use. Before use, the dialysis tube is filled with water and then drained, and the dialysis bag is thoroughly cleaned.

Preparation of gastric buffer: 2000mL of a mixed solution of 81mmol/L of sodium chloride, 6mmol/L of potassium chloride, 10mmol/L of hydrochloric acid and 2g/L of potassium sorbate (antibacterial agent) is prepared, and the pH of the solution is adjusted to 2.0 at 39 ℃ by using 2mol/L of hydrochloric acid.

Preparation of small intestine buffer: 2000mL of mixed solution with the concentration of 30mmol/L disodium hydrogen phosphate, the concentration of 170mmol/L sodium dihydrogen phosphate, the concentration of 90mmol/L sodium chloride, the concentration of 15mmol/L potassium chloride, the concentration of 800U/L penicillin and the concentration of 2g/L potassium sorbate is prepared. The pH of the solution was adjusted to 6.44 with 2mol/L phosphoric acid or 2mol/L sodium hydroxide at 39 ℃.

Simulated gastric fluid: the activity of pepsin in the reagent was determined according to the method for measuring the activity of pepsin. 223KU pepsin is weighed and dissolved in 250mL hydrochloric acid solution with the concentration of sodium chloride being 81mmol/L, the concentration of potassium chloride being 6mmol/L, the concentration of hydrochloric acid being 10mmol/L and the pH value being 2.0, and the solution is slowly stirred until the solution is dissolved, so that the pepsin activity concentration of the simulated gastric juice is 892U/mL.

Preparation of concentrated simulated small intestine solution: according to the method for measuring the activities of the alpha-amylase, the trypsin and the chymotrypsin, the activities of corresponding digestive enzymes in the reagent-grade amylase, the trypsin and the chymotrypsin are measured. Weighing and dissolving 60.9KU of alpha-amylase, 19.00KU of trypsin and 2.38KU of chymotrypsin in 25mL of deionized water, and slowly stirring until the alpha-amylase, the trypsin and the chymotrypsin are dissolved. So that the concentration of amylase is 2436U/mL, the concentration of trypsin is 760U/mL, and the concentration of chymotrypsin is 95U/mL.

Preparation of concentrated supplement simulated small intestine solution: according to the method for measuring the activities of the alpha-amylase, the trypsin and the chymotrypsin, the activities of corresponding digestive enzymes in the reagent-grade amylase, the trypsin and the chymotrypsin are measured. Weighing 30.5KU of alpha-amylase, 21.5KU of trypsin and 0.75KU of chymotrypsin, dissolving in 15mL of deionized water, and slowly stirring until dissolving. The concentration of amylase was 2037U/mL, the concentration of trypsin was 1430U/mL, and the concentration of chymotrypsin was 50U/mL.

Preparing: the gastric buffer solution and the small intestine buffer solution are repeatedly put into a buffer solution temperature control storage chamber of a monogastric animal bionic digestive system (model SDS-2 produced by Intelligent science and technology limited in Hunan, and the pipeline of the system is well connected with a buffer solution bottle. In the control software of the bionic digestion system, the preheating time of the bionic digestion system of the monogastric animals is set to be 60 min; the mixing frequency was set at 180 rpm; the flow rate of the buffer solution is set to be 60 mL/min; the temperatures of the digestion simulation chamber, the buffer solution control chamber and the pipeline heat preservation chamber are set to be 39 ℃; the temperature of the digestive juice heat preservation chamber is set to be 6 ℃; the stomach digestion time is set to be 1, 2, 3 and 4 hours; the emptying time of the gastric buffer solution is set to be 4 min; setting the volume of a cleaning solution (deionized water) at the gastric digestion stage to be 300 mL/repeated, setting the cleaning time to be 40min, and setting the cleaning times to be 3 times; the switching cycle time of the stomach-small intestine buffer solution is set for 30min, the injection volume of the concentrated simulated small intestine solution is set to be 2mL, the volume of the supplemented concentrated simulated small intestine solution is set to be 1mL, and the digestion time of the small intestine is set to be 0, 2, 4, 6, 8, 12 and 16 h; the injection time of the concentrated simulated small intestinal juice and the injection time of the supplemented concentrated simulated small intestinal juice are respectively set to be 0h and 4h of small intestinal digestion, and the emptying time of the buffer solution is set to be 4 min.

Loading: after the bionic digestion system finishes preheating, the treated dialysis tube longitudinally passes through the simulated digestion tube, the two ends of the dialysis tube are turned outwards, the dialysis tube is fastened by a rubber band, and the dialysis tube is fixed on the simulated digestion tube. Then, a plug was closed with a flap silica gel plug. Weighing 1-2 g of feed sample (2 g of compound feed and energy feed, 1g of protein and accurate to 0.0002g) into a glass test tube, pouring the sample in the glass test tube into a simulated digester provided with a dialysis tube, flushing the test tube with 20mL of simulated gastric juice, pouring into the dialysis tube, and plugging with a silica gel plug. The simulated digester is placed in a bionic digestion system of a monogastric animal, and a pipeline is connected according to the principle that water enters from the lower end of the simulated digester and water exits from the upper end of the simulated digester. Each group of 5 simulated digesters is connected in series. The digestive juice adding tube and the buffer solution are connected with the simulated digester through a quick joint. Clicking the starting option of the control software of the bionic digestion system of the monogastric animals to start the automatic operation of the bionic digestion process of the stomach and the small intestine of the pigs.

Bionic digestion: the bionic digestion temperature, time, buffer solution switching and residual liquid emptying of the stomach and small intestine stages are all automatically carried out according to the parameters set in the control software.

The following steps: after completion of digestion, undigested debris in the dialysis tubing was transferred without loss to a 50mL graduated cylinder and the volume read. Then, the cells were transferred to a 50mL centrifuge tube and centrifuged at 1250g at 4 ℃ for 10 minutes to obtain a supernatant.

4 method for measuring digestive enzyme Activity

The activity determination of pepsin, alpha-amylase, trypsin and chymotrypsin in the supernatant of the lower sample is a known technology, wherein: the activity of pepsin can be determined by reference to the relevant description in "Wirnt R and Wolf-Peter F.Pepsin,2.Assay at 280nm. In: Bergmayer H. methods of enzymic analysis [ M ]. Weinheinm: Verlag chemie.1974", Assay principle: the absorbance change of the amino acid content soluble in trichloroacetic acid solution generated by taking bovine hemoglobin as a substrate at 280nm has certain correlation with the activity of pepsin. The activity of alpha-amylase can be determined by reference to the description in the "Dahlqvist A.A method for the determination of the degree of amylase in the endogenous content. Scandinavian Journal of Clinical and Laboratory Investigation,1962,14: 145-151", which is based on the following assay principles: the reducing substance of the soluble starch after the action of amylase and 3, 5-dinitrosalicylic acid can form a colored complex, and the shade of the color of the complex is in a linear relation with the amount of the reducing substance at 530 nm; trypsin activity can be determined by reference to the relevant description In "Wirnt R.Trypsin, measurements with n α -p-toluenesulfonyl-l-argine methyl ester as substrate [ A ]. In, Bergmayer H U.methods of enzymic analysis, Weinheinm: Verlag chemistry.1974" on the principle of determination: the change speed of the absorbance of p-toluenesulfonic acid-L-arginine obtained by hydrolyzing the p-toluenesulfonic acid-L-arginine ethyl ester by trypsin at 247nm is the first-order speed of enzymatic reaction; the activity of chymotrypsin can be determined by reference to the relevant description in "Wirnt R.Chymotrypsin, measurements with n-benzyl-l-tyrosin ethyl ester as substrate. in: Bergmayer H. U.S. methods of enzymic analysis. Weinheinm: Verlag chemistry. 1974" on the principle of determination: the change of the absorbance of benzoyl-L-tyrosine ethyl ester hydrolyzed into benzoyl-L-tyrosine by chymotrypsin at 256nm is the first-order speed of enzymatic reaction.

5 change of digestive enzyme activity along with digestion time in pig stomach-small intestine bionic digestion process

5.1 variation of pepsin Activity with digestion time in Bionically digestive Process

TABLE 1 Change in pepsin Activity during gastric biomimetic digestion

When the simulated gastric fluid is mixed with corn, soybean meal, wheat bran or corn-soybean meal type feed, the specific activities of pepsin in the digestive fluid are respectively 59.1%, 28.8%, 30.3% and 32.7% of the corresponding activities of the simulated gastric fluid. At 1, 2, 3, 4h of simulated digestion, both the specific and total pepsin activity in the digestive juices was significantly higher than the corresponding pepsin activity in the digestive juices (P <0.05) when the sample was mixed with the simulated gastric juice just (time 0). When the simulated digestion of the corn is carried out for 1, 2 and 4 hours, the specific activity and the total activity of the pepsin are increased in a quadratic curve (P is less than 0.05), and the activity of the pepsin is stable within 3-4 hours. The specific activity and the total activity of the pepsin in the digestive juice at 4h reach 95.1 percent and 101.4 percent of the corresponding activity of simulated gastric juice. When the soybean meal is subjected to simulated digestion for 1 and 2 hours, the specific activity and the total activity of the pepsin are sequentially and remarkably increased (P is less than 0.05), the specific activity and the total activity of the pepsin are stable in 3-4 hours, but the specific activity is remarkably reduced (P is less than 0.05) compared with the specific activity in 2 hours, and the total activity is not remarkably different. Overall, the specific activity versus total activity of pepsin was plotted quadratic with simulated digestion time (P < 0.05). The specific activity and the total activity of the pepsin in the digestive juice at 4h reach 89.5 percent and 102.0 percent of the corresponding activity of simulated gastric juice. The difference in the specific activity of pepsin was not significant at 1, 2h of wheat bran digestion simulation, and the specific activity of pepsin was in a decreasing change at the subsequent 3, 4 h. While the total activity was not significantly different at

simulated digestion

1, 2, 3h, but significantly higher than the total activity of 4 h. Overall, the specific activity versus total activity of pepsin increased quadratically with simulated digestion time (P < 0.05). The specific activity and the total activity of the pepsin in the digestive juice at 4h reach 96.1 percent and 102.7 percent of the corresponding activity of simulated gastric juice. When the corn-soybean meal type diet is subjected to simulated digestion for 1, 2 and 3 hours, the specific activity and the total activity of the pepsin are sequentially and obviously increased (P is less than 0.05), and the activity of the pepsin is stable within 3-4 hours. Both the specific and total pepsin activities were plotted quadratically with simulated digestion time (P < 0.05). The specific activity and the total activity of the pepsin in the digestive juice at 4h reach 78.0 percent and 87.2 percent of the corresponding activity of simulated gastric juice.

5.2 variation of digestive enzyme Activity with digestion time in Small intestine Bionical digestion Process

FIG. 2 is a graph of the activity of digestive enzymes of corn in the simulated digestion stage of the small intestine as a function of digestion time, wherein the amylase activity concentration: SEM 1.21U/mL, variance P <0.01, linear mismatching P <0.01, quadratic P < 0.01; active concentration of trypsin: SEM is 0.25U/mL, variance P <0.01, linear mismatching P <0.01, quadratic P < 0.01; chymotrypsin activity concentration: SEM is 0.32U/mL, variance P <0.01, linear mismatching P <0.01, quadratic P < 0.01; total Amylase Activity: SEM ═ 0.20KU, variance P <0.01, linear mismatching P <0.01, quadratic P < 0.01; total trypsin activity: SEM ═ 0.04KU, variance P <0.01, linear mismatching P <0.01, quadratic P < 0.01; chymotrypsin total activity: SEM ═ 0.05KU, variance P <0.01, linear mismatching P <0.01, quadratic P < 0.01.

In the small intestine digestion stage of corn (see figure 2), the specific activity and the total activity of amylase in a digestion solution are reduced in a quadratic curve with digestion time (P <0.05), wherein the specific activity and the total activity are obviously reduced in 0-4 h (P < 0.05); 6-16 h are relatively smooth, but are all significantly lower than the corresponding values of 0 and 2 h. The specific activity and the total activity of the trypsin show a rapid decrease (P <0.05) within 0-8 h, and a quadratic curve change with a lower value is kept within 8-16 h. The specific activity and the total activity of chymotrypsin are changed in a quadratic curve, and reach the highest in 2h, and then are reduced remarkably (P < 0.05).

Fig. 3 is a graph showing the variation of the digestive enzyme activity of soybean meal with digestion time at the simulated digestion stage of the small intestine, wherein the amylase activity concentration: SEM 1.25U/mL, variance P <0.01, linear mismatching P <0.01, quadratic P0.51; active concentration of trypsin: SEM is 0.46U/mL, variance P <0.01, linear mismatching P <0.01, quadratic P < 0.01; chymotrypsin activity concentration: SEM is 0.37U/mL, variance P <0.01, linearity P < 0.01. Total Amylase Activity: SEM 0.19KU, variance P <0.01, linear mismatching P <0.01, quadratic P0.39; total trypsin activity: SEM ═ 0.07KU, variance P <0.01, linear mismatching P <0.01, quadratic P < 0.01; chymotrypsin total activity: SEM ═ 0.06KU, variance P <0.01, linear mismatching P <0.05, quadratic P < 0.01.

During the small intestine digestion stage of soybean meal (fig. 3), amylase specific activity and total activity in the digestive juice showed non-linear changes with digestion time. The difference between amylase specific activity and total activity at 0h and 4h was not significant, but significantly lower than the corresponding value of 2h (P < 0.05); the amylase specific activity and the total activity are sequentially and remarkably reduced (P is less than 0.05) within 4-12 h, and then the amylase tends to be stable. The specific activity and the total activity of the trypsin are obviously reduced in a quadratic curve along with the digestion time (P is less than 0.05), the activity is rapidly reduced in 0-8 h as a whole, and the activity of the trypsin is reduced to a lower level in 8-16 h. The specific activity and the total activity of the chymotrypsin are reduced in a quadratic curve, wherein the specific activity and the total activity are slowly reduced within 0-12 h, and are remarkably reduced within 12-16 h (P < 0.05).

FIG. 4 is a graph of wheat bran digestive enzyme activity versus digestion time at the simulated digestion stage of the small intestine; wherein, the amylase activity concentration is as follows: SEM 1.36U/mL, variance P <0.01, linear mismatching P <0.01, quadratic P < 0.01; active concentration of trypsin: SEM is 0.46U/mL, variance P <0.01, linear mismatching P <0.01, quadratic P < 0.01; chymotrypsin activity concentration: SEM is 0.30U/mL, variance P <0.01, linear mismatching P <0.01, quadratic P < 0.01. Total Amylase Activity: SEM ═ 0.21KU, variance P <0.01, linear mismatching P <0.01, quadratic P < 0.05; total trypsin activity: SEM ═ 0.07KU, variance P <0.01, linear mismatching P <0.01, quadratic P < 0.01; chymotrypsin total activity: SEM ═ 0.04KU, variance P <0.01, linear mismatching P <0.01, quadratic P < 0.01.

In the small intestine digestion stage of wheat bran (figure 4), the specific activity and the total activity of amylase in a digestive liquid are sequentially reduced in a quadratic curve along with the digestion time (P is less than 0.05), the activity is rapidly reduced within 0-8 hours, and the activity is slowly reduced within 8-12 hours. The specific activity and the total activity of the trypsin are obviously reduced in a quadratic curve along with the digestion time (P is less than 0.05), the activity is rapidly reduced in 0-8 h as a whole, and the activity of the trypsin is reduced to a lower level in 8-16 h. The specific activity and the total activity of the chymotrypsin are reduced in a quadratic curve, wherein the specific activity and the total activity are rapidly reduced within 0-12 hours and slowly reduced within 12-16 hours (P < 0.05).

Fig. 5 is a graph of the digestive enzyme activity of a corn-soybean meal type diet as a function of digestion time in the simulated digestion stage of the small intestine, wherein the amylase activity concentration: SEM 1.73U/mL, variance P <0.01, linear mismatching P0.07, quadratic P0.38; active concentration of trypsin: SEM is 0.39U/mL, variance P <0.01, linear mismatching P <0.05, quadratic P < 0.01; chymotrypsin activity concentration: SEM is 0.23U/mL, variance P <0.01, linear mismatching P <0.01, quadratic P < 0.01; total Amylase Activity: SEM 0.29KU, variance P <0.05, linear mismatching P <0.05, quadratic P0.62; total trypsin activity: SEM ═ 0.06KU, variance P <0.01, linear mismatching P <0.01, quadratic P < 0.01; chymotrypsin total activity: SEM ═ 0.04KU, variance P <0.01, linear mismatching P <0.01, quadratic P < 0.01.

In the small intestine digestion stage of the corn-soybean meal type diet (fig. 5), the specific activity and total activity of amylase show nonlinear changes with time digestion, wherein the specific activity between 4-16 h has no significant difference, and the specific activity of only 0 and 2h is significantly higher than that of 4 and 16h (P < 0.05); the difference in total activity was not significant for the other digestion times except 4 h. The specific activity and the total activity of the trypsin are obviously reduced in a quadratic curve along with the digestion time (P is less than 0.05), the activity is rapidly reduced in 0-12 hours on the whole, and the activity is slowly increased in 12-16 hours. The specific activity of the chymotrypsin is the highest in 0-4 h, the chymotrypsin is remarkably reduced (P <0.05) in 6-12 h, the chymotrypsin is remarkably increased in 0-4 h before the total activity of the chymotrypsin, and then is remarkably reduced (P <0.05), and the chymotrypsin overall shows a quadratic curve change.

6 effect of supplementing digestive enzymes in small intestine digestion stage in bionic digestion process of pig

The activities of amylase, trypsin and chymotrypsin in the bionic digestion small intestine stage of the corn, soybean meal, wheat bran and corn-soybean meal type feed are attenuated in different degrees along with time, and the change condition of the enzyme in the simulated small intestine liquid after the digestive enzyme is supplemented in the small intestine stage for 4 hours is listed in table 2. After the corn is compensated with digestive enzymes in the small intestine digestion stage, the amylase specific activity and the total activity are remarkably higher than corresponding values of 6-8 h (P <0.05) in 4h and higher than initial values (150.8U/mL), wherein the specific activity of 4h is closer to the in-vivo value (221U/mL) of the pig. The specific activity and total activity of trypsin still showed a sharp decline of the change law (P <0.05), with the specific activity of 4h higher than the initial value, closer to the in vivo value (69.1U/mL). The specific activity and the total activity of the chymotrypsin are remarkably higher than corresponding values (P <0.05) of 6-8 h in 4h, and are closer to the initial values on the whole. After the soybean meal compensates digestive enzymes in the small intestine stage, the difference of the specific activity and the total activity of the amylase is not obvious (P is more than 0.05), and the specific activity and the total activity of the amylase are both higher than the initial value (145.3U/mL) and are closer to the in vivo value (221U/mL) of the pig. The specific activity and the total activity of the trypsin still show a sharp decline change rule (P <0.05), wherein the specific activity of 4h and the total activity are closer to the initial value. The specific activity and the total activity of chymotrypsin show a change of increasing and decreasing overall, which is generally close to the initial values. After wheat bran was used to compensate for digestive enzymes at the small intestine stage, the amylase specific activity and the total activity differed insignificantly (P > 0.05), but slightly below the initial and in vivo values. The specific activity and the total activity of trypsin still showed a sharp decreasing change law (P <0.05), with the specific activity of 4h being closer to the initial value. The difference between the specific activity and the total activity of the chymotrypsin is not obvious within 4-6 h, but is obviously higher than the corresponding value (P <0.05) of 6h, and both are lower than the initial value. After the corn-soybean meal type diet was compensated for digestive enzymes at the small intestine stage, the amylase specific activity and total activity differed insignificantly between 4h and 8h, but was higher than the corresponding value of 6h (P < 0.05). Wherein the specific activity of 4h is higher than the initial value and is closer to the in vivo value of the pig. The specific activity and the total activity of trypsin still showed a sharp decreasing change law (P <0.05), with the specific activity of 4h being closer to the initial value. The specific activity of the chymotrypsin is remarkably higher than the corresponding value (P <0.05) of 6-8 h in 4h, and is closer to the initial value as a whole, and the difference of the total activity among digestion times is not remarkable.

TABLE 2 bionic digestion of the small intestine for 4h to compensate for the changes in the activity of amylase, trypsin, chymotrypsin after digestion

Combining the above test results, the simulated gastric juice and the feed show a sharp decrease in activity at the stage of gastric digestion, but the pepsin activity can be restored to the range of in vivo values after 1h of digestion and remain relatively stable. Therefore, pepsin does not need to be supplemented in the bionic digestion of the pig; in the small intestine digestion simulation stage, the three main digestive enzymes are attenuated along with the digestion time, particularly the activity of trypsin is rapidly reduced, and after the simulated small intestine liquid is supplemented, concentrated and supplemented, the activity change of the 3 digestive enzymes can present the periodic change similar to the activity of the digestive enzymes in the porcine pancreatic liquid and the intestinal tract along with the meal, thereby more realistically simulating the in-vivo digestion process of the pig.

EXAMPLE two Effect of measuring digestibility of proteins of raw materials of conventional pig feed by biomimetic digestion

1 pretreatment of feed raw materials

The common pig feed raw materials of corn, soybean meal, wheat bran and cottonseed meal are randomly selected as representative samples. After being respectively crushed by a universal crusher and passing through a standard sieve hole of 60 meshes, the materials are vacuumized, filled with nitrogen and packaged by sample bags according to the embodiment 1 and then stored in a freezer at the temperature of 15 ℃ below zero for standby.

Bionic digestion method of pig

Preparation of concentrated simulated small intestine solution: according to the method for measuring the activities of the alpha-amylase, the trypsin and the chymotrypsin, the activities of corresponding digestive enzymes in the reagent-grade amylase, the trypsin and the chymotrypsin are measured. The concentration of alpha-amylase in the prepared concentrated simulated small intestine solution is 2436U/mL, the concentration of trypsin is 760U/mL, and the concentration of chymotrypsin is 95U/mL.

Preparation of concentrated supplement simulated small intestine solution: according to the method for measuring the activities of the alpha-amylase, the trypsin and the chymotrypsin, the activities of corresponding digestive enzymes in the reagent-grade amylase, the trypsin and the chymotrypsin are measured. The concentration of alpha-amylase in the prepared concentrated supplementary simulated small intestinal juice is 2037U/mL, the concentration of trypsin is 1430U/mL, and the concentration of chymotrypsin is 50U/mL.

Preparing: the gastric buffer solution and the small intestine buffer solution are repeatedly put into a buffer solution temperature control storage chamber of a monogastric animal bionic digestive system (model SDS-2 produced by Intelligent science and technology limited in Hunan, and the pipeline of the system is well connected with a buffer solution bottle. In the control software of the bionic digestion system, the preheating time of the bionic digestion system of the monogastric animals is set to be 60 min; the mixing frequency was set at 180 rpm; the flow rate of the buffer solution is set to be 60 mL/min; the temperatures of the digestion simulation chamber, the buffer solution control chamber and the pipeline heat preservation chamber are set to be 39 ℃; the temperature of the digestive juice heat preservation chamber is set to be 6 ℃; setting the gastric digestion time for 4 hours; the emptying time of the gastric buffer solution is set to be 4 min; setting the volume of a cleaning solution (deionized water) at the gastric digestion stage to be 300 mL/repeated, setting the cleaning time to be 40min, and setting the cleaning times to be 3 times; setting 30min at the early stage of small intestine digestion, setting the injection volume of the concentrated simulated small intestine liquid to be 2mL, setting the volume of the supplemented concentrated simulated small intestine liquid to be 1mL, and setting the digestion time of the small intestine to be 8 h; the injection time of the concentrated simulated small intestinal juice and the injection time of the supplemented concentrated simulated small intestinal juice are respectively set to be 0h and 4h of small intestinal digestion, and the emptying time of the buffer solution is set to be 4 min. The volume of the cleaning solution used for final cleaning is set to be 300 mL/repetition, the cleaning time is set to be 120min, and the cleaning times are set to be 6 times

Loading: after the bionic digestion system finishes preheating, the treated dialysis tube longitudinally passes through the simulated digestion tube, the two ends of the dialysis tube are turned outwards, the dialysis tube is fastened by a rubber band, and the dialysis tube is fixed on the simulated digestion tube. Then, a plug was closed with a flap silica gel plug. Weighing 1-2 g of feed sample (2 g of compound feed and energy feed, 1g of protein and accurate to 0.0002g) into a glass test tube, pouring the sample in the glass test tube into a simulated digester provided with a dialysis tube, flushing the test tube with 20mL of simulated gastric juice, pouring into the dialysis tube, and plugging with a silica gel plug. The simulated digester is placed in a bionic digestion system of a monogastric animal, and a pipeline is connected according to the principle that water enters from the lower end of the simulated digester and water exits from the upper end of the simulated digester. Each group of 5 simulated digesters is connected in series. The digestive juice adding tube and the buffer solution are connected with the simulated digester through a quick joint. Clicking the starting option of the bionic digestive system of the monogastric animal to start the automatic operation of the bionic digestive process of the pig stomach and small intestine.

Bionic digestion: the bionic digestion temperature, time, buffer solution switching, residual liquid emptying and hydrolysate cleaning at the stomach and small intestine stages are all automatically carried out according to the parameters set in the control software.

The following steps: after digestion, the undigested residue in the dialysis tubing was transferred without loss to a petri dish of known absolute dry weight, dried at 65 ℃ until no water mark was formed (optionally freeze-dried), and then dried at 105 ℃ to constant weight.

The crude protein content of the feed sample and the undigested residue were measured separately,

the protein digestibility ═ 100 (feed crude protein content × sample weight-undigested residue crude protein content × undigested residue weight + digestive enzyme residual protein content)/(feed crude protein content × sample weight).

3 bionic digestion method for determining protein digestibility of feed raw materials

TABLE 3 determination of protein digestibility of feed raw materials by biomimetic digestion and crude protein digestion of ileum terminal of growing pig by in vivo method

Comparison of rates

Among The 4 commonly used pig feed materials, corn and two protein feed materials (> 40% crude protein content) -soybean meal and cottonseed meal biomimetic digestion methods found protein digestibility to closely match The literature reports (Sauvant, D., Perez, J., M., Tran, G.,2004.Tables of composition and nutritional value of materials. Generation Academic publications and INRA Editions; National Research study of pig. 2012. Nutrition Requirements of Swine:10th Revised edition Washington, DC: The National academy Press) growing pig standard ileal end digestibility. The crude protein digestibility of the wheat bran bionic digestion method is slightly higher than the standard ileum terminal digestibility reported in the literature and still falls within the range of sample variation, which indicates that the crude protein digestibility determined by the method can represent the measured value of animal experiments. In addition, the variation coefficients of the crude protein digestibility measured by the bionic digestion method are all within 1 percent, which shows that the method has very high precision.

The above-mentioned preferred embodiments of the present invention and the technical principles applied thereto are obvious to those skilled in the art that any equivalent changes, simple substitutions and the like based on the technical solutions of the present invention are within the protection scope of the present invention without departing from the spirit and scope of the present invention.

Claims

1. A method for determining the digestibility of pig feed protein by in vitro biomimetic digestion comprises the following steps:

4) preparation of simulated small intestine solution: according to the activity of digestive enzyme in the porcine intestinal fluid, preparing concentrated simulated intestinal fluid by using reagent-grade alpha-amylase, trypsin and chymotrypsin; preparing concentrated supplementary simulated intestinal juice by using reagent-grade alpha-amylase, trypsin and chymotrypsin;

6) gastric simulated digestion: pumping the gastric buffer solution out of a dialysis tube of the monogastric animal simulated digester and then returning the gastric buffer solution into a gastric buffer solution reagent bottle for reciprocating circulation, and providing hybrid power of simulated gastric juice and a feed sample through rotary oscillation to perform simulated digestion of the stomach; discharging the gastric buffer solution in the simulated digester after digestion is finished, and pumping deionized water into the gastric buffer solution to clean hydrolysate remained in the dialysis tube in the simulated digestion stage of the stomach; discharging the cleaning liquid in the simulated digester after the cleaning is finished;

8) cleaning of hydrolysate: pumping deionized water out of a dialysis tube of the monogastric animal simulated digester to clean hydrolysate remained in the dialysis tube in the whole simulated digestion stage; discharging the cleaning liquid in the simulated digester after the cleaning is finished;

9) analysis of undigested residue and calculation of digestibility: transferring the residue in the dialysis tube in the step 8) into a culture dish, drying, respectively measuring the content of crude protein or amino acid in the residue and the pig feed sample, and calculating the digestibility of the crude protein or amino acid;

in the step 2), the concentration of hydrochloric acid in the gastric buffer solution is 5-10 mmol/L, the concentration of NaCl is 60-110 mmol/L, the concentration of KCl is 4-8 mmol/L, and the concentration of an antibacterial agent is 1-2 g/L; adjusting the pH value to 2-3 at 39 ℃; the antibacterial agent is potassium sorbate;

the concentration of disodium hydrogen phosphate in the small intestine buffer solution is 30mmol/L, the concentration of sodium dihydrogen phosphate is 170mmol/L, the concentration of NaCl is 70-106 mmol/L, the concentration of KCl is 14-17 mmol/L, the concentration of penicillin is 800U/L, and the concentration of an antibacterial agent is 1-3 g/L; adjusting the pH value to 6.4-6.5 by using 1-2 mol/L phosphoric acid or sodium hydroxide at 39 ℃; the antibacterial agent is potassium sorbate;

in the step 3), the activity of pepsin in the simulated gastric juice is 737-1000U/mL, the concentration of hydrochloric acid is 5-10 mmol/L mol/L, the concentration of NaCl is 60-110 mmol/L, the concentration of KCl is 4-8 mmol/L, and the pH value is 2-3;

in the step 4), the concentration of amylase in the concentrated simulated small intestine solution is 2046-2893U/mL, the concentration of trypsin is 627-858U/mL, and the concentration of chymotrypsin is 77-110U/mL;

2. The method of claim 1, wherein: in the step 1), the pig feed is a single feed raw material or a compound feed;

the crushing is carried out in a universal crusher; when the feed raw materials are crushed into 2mm sieve pieces in production, crushing the samples in a universal crusher for 20-30 s, and sieving the samples with a 40-mesh grading sieve; when the feed raw materials are crushed and sieved by a sieve sheet with the thickness of 1mm in production, the sample is crushed in a universal crusher for 60-90 s and sieved by a 60-mesh classifying sieve;

in the step 1), the method for performing vacuum-nitrogen treatment on the crushed sample specifically comprises the following steps: filling the crushed sample into a breathable cotton paper bag, sealing the bag, then sleeving the bag into a plastic sample bag, adjusting the air suction time to be 10-20 s, the air inflation time to be 2-4 s and the heating sealing time to be 1.5-4.5 s on a vacuum packaging machine, and then carrying out vacuumizing-nitrogen filling-sealing treatment; the low-temperature storage temperature is-20 to-10 ℃.

3. The method of claim 1, wherein: in the step 5), the cut-off molecular weight of the dialysis tube is 12000-14400 daltons, and the flat diameter is 44 mm; the opening volume of the tube body is 35-45 mL.

4. The method of claim 1, wherein: in the step 5), the monogastric bionic digestion system is a monogastric bionic digestion system of model SDS-2 produced by Intelligent science and technology limited in Hunan;

5. The method of claim 1, wherein: in the step 6), the digestion temperature is 39 ℃; the rotary oscillation frequency of the feed sample mixed with the simulated gastric juice is 180 revolutions per minute; the flow rate of the buffer solution flowing through the simulated digester was 60 mL/min; the simulated digestion time of the stomach is 4 hours; after the simulated digestion of the stomach is finished, the cleaning liquid amount for cleaning the digestion product at one time is 300mL of deionized water/simulated digester, each time of cleaning is 40 minutes, and the cleaning is repeated for 3 times;

6. The method of claim 1, wherein: in the step 8), the cleaning liquid amount for cleaning the digestion product at one time after the whole simulated digestion is finished is 300mL of deionized water/simulated digester, the cleaning is carried out for 2-4 h every time, and the cleaning is repeated for 6 times.

7. The method of claim 1, wherein: in the step 9), the undigested residues are transferred into a culture dish with constant weight, and then are dried in a constant temperature forced air drying oven at 65 ℃ until no water mark exists, or are freeze-dried, and then are dried in a constant temperature forced air drying oven at 105 ℃ until the weight is constant;

measuring the content of crude protein in the feed sample and undigested residues by using a Kjeldahl method, and calculating the digestibility of the protein; or measuring the content of amino acid by liquid chromatography or full-automatic amino acid analyzer, and calculating the digestibility of amino acid.